The transient receptor potential vanilloid 1 (TRPV1) is a polymodal ion channel essential to the cellular mechanism underlying the detection of noxious stimuli. TRPV1 is activated by heat, protons, capsaicin, and animal toxins, and is modulated by proalgesic inflammatory agents (e.g., bradykinin, bioactive lipids) produced in response to tissue injury. Our long-term goal is to delineate the roles of polymodal ion channels in sensory neuron excitation and the mechanisms by which they contribute to inflammatory pain. The rationale for our proposed research is that a deeper mechanistic understanding of TRPV1 proton- and heat-dependent gating would greatly facilitate the development of strategies to ameliorate TRPV1-mediated inflammatory pain, without disrupting normal sensory physiology. While functional and structural characterization of TRPV1 have shed light on the mechanisms of capsaicin and toxin activation, the processes whereby the two main endogenous activators, protons and heat, trigger gating remain largely unknown. Moreover, the intracellular TRPV1 C terminus is a key regulatory site for regulating stimulus sensitivity. However, any potential allosteric interacting regions or putative contacts with the plasma membrane have yet remain to be explored. It is our contention that spectroscopic approaches are needed to fully define the allosteric conformational changes responsible for TRPV1 activation and to depict the C-terminal/membrane interaction. To this end, we will carry out electrophysiological analyses together with electron paramagnetic resonance spectroscopy experiments in both closed and open states. With these data, we will depict the conformational changes that TRPV1 undergoes during proton- and heat-dependent gating. We will pursue two Specific Aims: 1) Determine the dynamic conformational rearrangements of TRPV1 during proton and heat activation, and 2) Explore the interaction between the TRPV1 C-terminal domain and the plasma membrane. The proposed research is significant because it is expected to have broad translational importance in the treatment of pain associated with a wide range of pathophysiological conditions.